Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer-related mortality, with a five year survival less than 5%. The overwhelming majority of patients succumb to metastatic disease or tumor recurrence following apparently curative resection. Current treatment efforts in PDAC are focused almost entirely on reduction in bulk tumor size, rather than elimination of cells with tumor initiating and metastatic abilities. Research performed in our laboratory over the last funding cycle has identified a putative cancer stem cell (CSC) compartment in PDAC, delineated by high levels of expression of the enzyme aldehyde dehydrogenase. This minor subpopulation of ALDH bright cells is capable of self-renewal and tumor initiation, and is observed in elevated numbers in PDAC metastases. The ALDH bright cells are exquisitely dependent upon sustained Hedgehog (Hh) and Notch signals for their viability. Preferential elimination of the ALDH bright cells with small molecule Hh antagonists blocks systemic metastases and increases survival in PDAC preclinical models; conversely, gemcitabine enhances the relative proportion of ALDH bright cells, underscoring the pitfall of conventional anti-metabolite therapy. The current proposal will build upon these themes, by developing therapies that target the cellular compartment responsible for disease progression. In Aim 1, we propose the first Phase II clinical trial of an orally bioavailable Hh antagonist in combination with gemcitabine in patients with advanced PDAC, with the primary objective of improving overall survival. In Aim 2, we will study the emerging contribution Hh-dependent stromal responses to PDAC disease initiation and progression, including stromal interactions with the epithelial CSC compartment, using a newly developed conditional mouse model of Hh ligand misexpression. In Aim 3, we will initiate a comprehensive preclinical trial with an orally bioavailable inhibitor of Notch signaling in spontaneously metastatic orthotopic xenograft model of PDAC, as a prelude to future clinical application. We anticipate that developing parallel and complementary therapeutic approaches against two critical developmental signaling pathways that sustain CSC viability in PDAC provides an unprecedented opportunity for ameliorating the dismal prognosis of this malignancy.